Oxidation-reduction properties of the regulatory site of spinach phosphoribulokinase.
The oxidation-reduction midpoint potential (Em) of the regulatory disulfide, formed between Cys16 and Cys55, of spinach chloroplast phosphoribulokinase has been determined both for the wild-type enzyme and for a C244S-C250S double mutant, using enzymatic activity to monitor the oxidation-reduction state of the regulatory disulfide. At pH 7.0, Em values for the two-electron reduction of the regulatory disulfide of -295 +/- 10 and -290 +/- 10 mV were measured for the wild-type and mutant, respectively. In contrast to the dependence of activity on ambient potential (Eh) observed for the wild-type enzyme and the double mutant, which both followed the Nernst equation for a two-electron process, high and constant activity was exhibited by a C16S-C244S-C250 triple mutant of the enzyme at all Eh values tested. Em values for the wild-type enzyme were also measured at pH values of 6.7, 7.5, 7.7, and 8.2 and the Em vs pH data in this region give a good fit to a straight line with a slope of -60 mV/pH unit.
Hirasawa
M
M
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409-1061, USA.
Brandes
H K
HK
Hartman
F C
FC
Knaff
D B
DB
D002736:Chloroplasts / Q000201:enzymology*; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010084:Oxidation-Reduction; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011199:Potentiometry; D018724:Spinacia oleracea / Q000201:enzymology
D004220:Disulfides; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D003545:Cysteine